Five near-infrared-emissive graphene quantum dots for multiplex bioimaging

Due to high tissue penetration depth and low autofluorescence backgrounds, near-infrared (NIR) fluorescence imaging has recently become an advantageous diagnostic technique used in a variety of fields. However, most of the NIR fluorophores do not have therapeutic delivery capabilities, exhibit low photostabilities, and raise toxicity concerns. To address these issues, we developed and tested five types of biocompatible graphene quantum dots (GQDs) exhibiting spectrally-separated fluorescence in the NIR range of 928-1053 nm with NIR excitation. Their optical properties in the NIR are attributed to either rare-earth metal dopants (Ho-NGQDs, Yb-NGQDs, Nd-NGQDs) or defect-states (nitrogen doped GQDS (NGQDs), reduced graphene oxides) as verified by Hartree-Fock calculations. Moderate up to 1.34% quantum yields of these GQDs are well-compensated by their remarkable >4 h photostability. At the biocompatible concentrations of up to 0.5-2 mg ml^-1 GQDs successfully internalize into HEK-293 cells and enable in vitro imaging in the visible and NIR. Tested all together in HEK-293 cells five GQD types enable simultaneous multiplex imaging in the NIR-I and NIR-II shown for the first time in this work for GQD platforms. Substantial photostability, spectrally-separated NIR emission, and high biocompatibility of five GQD types developed here suggest their promising potential in multianalyte testing and multiwavelength bioimaging of combination therapies.